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Martijn Derks Masoed Ramuz Nick Alberts Rico Hagelaar

The development of a RNA-sequencing pipeline based on tuxedo tools. Martijn Derks Masoed Ramuz Nick Alberts Rico Hagelaar. Index . Dataset Pipeline 1 ( Tophat_cuff ) Pipeline 2 ( Cuff_diff ) Pipeline 3 (Summary) Conclusions Future prospects. Dataset.

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Martijn Derks Masoed Ramuz Nick Alberts Rico Hagelaar

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  1. The development of a RNA-sequencing pipeline based on tuxedo tools Martijn Derks Masoed Ramuz Nick Alberts Rico Hagelaar

  2. Index • Dataset • Pipeline 1 (Tophat_cuff) • Pipeline 2 (Cuff_diff) • Pipeline 3 (Summary) • Conclusions • Future prospects

  3. Dataset • Arabidopsis thaliana (advanced) • Six conditions: • Cold stress • Drought stress • Heat stress • Highlight stress • Salt stress • Control Ganet al. 2011. Multiple reference genomes and transcriptomes for Arabidopsis thaliana. Nature. 477, P 419–423.

  4. Configuration file Input data (FastQ) Tophat_cuff Tophat (6x ) Bamfile Cufflinks Transcripts.gtf Analysis Total intron length Basic for plot R Transcript length

  5. Tophat_cuff results

  6. Tophat_cuff results

  7. Cuff_diff (1) (5x ) Control vs condition Cuffmerge transcript.gtf Merged.gtf Cuffdiff Bamfile DE-genes Functions + enrichment

  8. Cuff_diff (2) (5x ) uniprot Get Functions David Enrichment

  9. Cuff_diff results (Uniprot) • XLOC_005119 XLOC_005119 Hsp70b 1:5502205-5504535 WT_controlheat_stress OK 1.88554 4668.1 11.2736 -4.26394 2.00852e-05 0.00596 • 568 yesQ9S9N1 Heat shock 70 kDa protein 5 (Heat shock protein 70-5) (AtHsp70-5) (Heat shock protein 70b) FUNCTION: In cooperation with other chaperones, • Hsp70s stabilize preexistent proteins against aggregation and mediate the folding of newly translated polypeptides in the cytosol as well as within organelles. These • chaperones participate in all these processes through their ability to recognize nonnative conformations of other proteins. They bind extended peptide segments with a • net hydrophobic character exposed by polypeptides during translation and membrane translocation, or following stress-induced damage (By similarity). Cytopla • sm. ATP binding; cell wall; chloroplast; plasma membrane; response to heat; response to virus GO:0005524; GO:0005618; GO:0009507; GO:0005886; GO:0009408; GO: • 0009615

  10. Cuff_diff results DE genes/overlap

  11. Cuff_diff results (David) Cold Drought Heat Salt Highlight

  12. Summary Summary Tophat count AT_codes Csv maker GC genes vs FPKM CV Expr. intron Conservation Overlap matrix Clustering R CV= STDEV/ Average

  13. HC sample Clustering

  14. HC gene Clustering 0.15

  15. Heatmap Clustering

  16. HC clusters (9)

  17. PAM clusters (10)

  18. Transcription factors Abscisic acid biosynthesis (stress conditions) 1 2 1. Cold, salinity and drought stresses: An overview ShilpiMahajan NarendraTuteja 2. Cold stress regulation of gene expression in plants ViswanathanChinnusamy et al.

  19. Conserved genes in Arabidopsis • Abiotic stress genes which also occur in Arabidopsis were retrieved from Oryzasativa (Rabbani et al). • These genes were compared with the DE stress genes found in the results. • Three genes were found in the salt, cold and drought conditions. • Rabbani, M.A. Maruyama, K. Abe, H. Khan, M. A. Katsura, K. Ito, Yoshiwara, K. Seki, M. Shinozaki, K. Yamaguchi-Shinozaki, K. 2003. Monitoring Expression Profiles of Rice Genes under Cold, Drought, and High-Salinity Stresses and Abscisic Acid Application Using cDNA Microarray and RNA Gel-Blot Analyses. Plant Physiology vol. 133. No 4. Pp 1755-1767

  20. Literature overlap • Results of the GO enrichment are backed up by the literature, with the exception of high light stress • The crosstalk between drought, cold and salt stress was confirmed by the literature with a greater emphasis on drought and salt stress. Seki, M. Narusaka, M. Ishida, J. Nanjo, T. Fujita, M. Oono, Y. Kamiya, A. Nakajima, M. Enju, A. Sakurai, T. Satou, M. Akiyama, K. Taji, T. Yamaguchi-Shinozaki, K. Carninci, P. Kawai, J. Hayashizaki, Y. Shinozaki, K. 2002. Monitoring the expression profiles of 7000 Arabidopsis genes under drought, cold and high-salinity stresses using a full-length cDNA microarray. V 31. I 3. pp 279-292. Baniwal, K. S. Bharti, K. Yu Chan, K. Fauth, M. Ganguli, A. Kotak, S. Mishra, S. K. Nover, L. Port, M. Scharf, K. Tripp, J. Weber, C. Zielinski, D. Koskull-Doring, P. 2004. Heat stress response in plants: a complex game with chaperones and more than twenty heat stress transcription factors. J Biosci. V 29. I 4. pp471-487. Bartels, D. Nelson, D. 1994. Approaches to improve stress tolerance using molecular genetics. Plant, Cell and Environment. V 17. pp 659-667. Wang, W. Vinocur, B. Shoseyov, O. Altman, A. 2004. Role of plant heat-shock proteins and molecular chaperones in the abiotic stress response. V 9. I 5. pp. 244-252.

  21. Conclusions • Working pipeline for (Paired + Unpaired) RNAseq analysis • DE genes + Gene Enrichment detection • Cluster analysis CV genes • Differential expressed genes identified (stress conditions vs. WT) • Correlation Transcript length with FPKM • Not found in Intron/GC percentage • Clusters of Co-expressed genes • Assumption of co-regulated genes

  22. Future perspectives • Use different IDs (TAIR IDs are not suitable) • Transcription factors to cluster genes (similar regulatory elements? ) • Conservation other plant species (synteny) • Validation different dataset (organisms, paired end)

  23. Questions

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